Optically Active N-(Alpha-Mercaptopropionyl)Glycine

ABSTRACT

An optically active N-(α-mercaptopropionyl)glycine, i.e., R-(−)-N-(α-mercaptopropionyl)glycine or S-(−)-N-(α-mercaptopropionyl)glycine, a preparation method thereof, a pharmaceutical preparation containing the compound or a pharmaceutically acceptable salt or ester thereof, and use of the same in preparation of detoxification medicament for improving metabolism, are provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an optically activeN-(α-mercaptopropionyl)glycine, preparation method thereof, use of thesame in preparation of detoxification medicament for improvingmetabolism, and pharmaceutical preparation containing the same.

2. Related Art

N-(α-mercaptopropionyl)glycine, generic name tiopronin, is used totreat, for example, acute and chronic hepatitis, and cirrhosis inclinics, with very excellent therapeutic use in treatment of liverdiseases, and preparation method and pharmaceutical preparation thereofhave been frequently disclosed. A tiopronin pharmaceutical preparationis disclosed in Chinese Patent Application No. CN02129300.7, and aprocess for synthesizing tiopronin is disclosed in Chinese Journal ofMedicinal Chemistry (Vol. 7, No. 1, p. 55-56, March 1997). However,there is not any report for the optical activity, i.e., levo and dextroisomers of N-(α-mercaptopropionyl)glycine. For the majority of thedrugs, a single enantiomer has high therapeutic efficacy and low adverseeffect, and thus is called as eutomer, while the inactive or low-activeenantiomer is referred to as distomer; and in most cases, the distomerhas no therapeutic efficacy, but can also partly neutralize the effectof the enantiomer, and even incur serious adverse effect. Theenantiomeric drugs have very different pharmacologic activity andmetabolic process in the body, and thus exhibit different biologicalactivity and efficacy. In clinical application, due to the lack ofawareness of the differences in pharmacodynamic and pharmacokineticbehaviors of the individual enantiomer of chiral drugs, the conclusionssometimes contradict the therapeutic efficacy or occurrence of adverseeffect, and will even improperly direct the application of drugs inclinics. Therefore, researches on optical activities of drugs are verynecessary.

SUMMARY OF THE INVENTION

The present invention is directed to an optically activeN-(α-mercaptopropionyl)glycine.

The present invention is further directed to a method for preparing theoptically active N-(α-mercaptopropionyl)glycine.

The present invention is further directed to a use of the opticallyactive N-(α-mercaptopropionyl)glycine in preparation of detoxificationmedicament for improving metabolism.

The present invention is further directed to a pharmaceuticalpreparation containing the optically activeN-(α-mercaptopropionyl)glycine.

As embodied and broadly described herein, the present invention providesan optically active N-(α-mercaptopropionyl)glycine, which is levo ordextro N-(α-mercaptopropionyl)glycine.

The levo N-(α-mercaptopropionyl)glycine isR-(−)-N-(α-mercaptopropionyl)glycine

of R-configuration, or a pharmaceutically acceptable salt or esterthereof, in which the salt can be an amino acid salt or a metal salt.

The amino acid salt has a general structural formula below:

in which R can be an amino acid selected from, for example, arginine,lysine, glycine, aspartic acid, alanine, phenylalanine, leucine,isoleucine, ornithine, cystine, cysteine, tyrosine, valine, serine,histidine, threonine, tryptoophan, methionine, methionine, proline,glutamic acid, and hydroxyproline.

The metal salt has a general structural formula below:

in which R₀ can be, for example, potassium or sodium.

The ester has a general structural formula below:

in which R₁ is a linear C₁-C₅ alkyl group.

The dextro N-(α-mercaptopropionyl)glycine isS-(+)-N-(α-mercaptopropionyl)glycine

of S-configuration, or a pharmaceutically acceptable salt or esterthereof, in which the salt can be an amino acid salt or a metal salt.

The amino acid salt has a general structural formula below:

in which R can be an amino acid selected from, for example, arginine,lysine, glycine, aspartic acid, alanine, phenylalanine, leucine,isoleucine, ornithine, cystine, cysteine, tyrosine, valine, serine,histidine, threonine, tryptoophan, methionine, methionine, proline,glutamic acid, and hydroxyproline.

The metal salt has a general structural formula below:

in which R₀ can be, for example, potassium or sodium.

The ester has a general structural formula below:

in which R₁ is a linear C₁-C₅ alkyl group.

The present invention also provides a method for preparing the opticallyactive N-(α-mercaptopropionyl)glycine, which includes a method forpreparing R-(−)-N-(α-mercaptopropionyl)glycine and a method forpreparing S-(−)-N-(α-mercaptopropionyl)glycine.

I. The method for preparing R-(−)-N-(α-mercaptopropionyl)glycineincludes:

1. reacting R-(+)-2-chloropropionic acid with thionyl chloride to getR-(+)-2-chloropropionyl chloride;

2. reacting R-(+)-2-chloropropionyl chloride with glycine under a weakbasic condition to get R-(+)-2-chloropropionyl glycine; and

3. reacting sodium sulfide with sublimed sulfur to get sodium disulfide,which is then acidified by reacting with R-(+)-2-chloropropionylglycine, to get R-(−)-N-(α-mercaptopropionyl)glycine;

where the resulting R-(−)-N-(α-mercaptopropionyl)glycine is furtherreacted with an acid, a base, or an alcohol to get a salt or an ester.

II. The method for preparing S-(+)-N-(α-mercaptopropionyl)glycineincludes:

1. reacting S-(−)-2-chloropropionic acid with thionyl chloride to getS-(−)-2-chloropropionyl chloride;

2. reacting S-(−)-2-chloropropionyl chloride with glycine under a weakbasic condition to get S-(−)-2-chloropropionyl glycine; and

3. reacting sodium sulfide with sublimed sulfur to get sodium disulfide,which is then acidified by reacting with S-(−)-2-chloropropionylglycine, to get S-(+)-N-(α-mercaptopropionyl)glycine;

where the resulting S-(+)-N-(α-mercaptopropionyl)glycine is furtherreacted with an acid, a base, or an alcohol to get a salt or an ester.

The present invention also provides a use of the optically activeN-(α-mercaptopropionyl)glycine, i.e.,R-(−)-N-(α-mercaptopropionyl)glycine orS-(+)-N-(α-mercaptopropionyl)glycine, or a pharmaceutically acceptablesalt or ester thereof in preparation of detoxification medicament forimproving metabolism, and particularly a use in treatment of acute andchronic liver diseases and improvement of liver function, includingprotection of liver tissue cells, treatment of various hepatitis, forexample, acute and chronic hepatitis, viral hepatitis, alcoholichepatitis, drug induced hepatitis, and heavy metal toxic hepatitis, andtreatment of fatty liver, acute and chronic liver injury, and cirrhosis;as well as in prevention and cure of peripheral blood leukopenia causedby chemoradiation therapy and accelerating of restoration of livercells, to reduce the adverse effect of chemotherapy; in prevention andcure of early senile cataract and vitreous opacities; and in heavy metaldetoxification.

It is found from pharmacodynamic experiment that bothR-(−)-N-(α-mercaptopropionyl)glycine andS-(+)-N-(α-mercaptopropionyl)glycine have good protection effect forliver injury, which is better than that ofN-(α-mercaptopropionyl)glycine.

It is found from pharmacokinetic experiment that, conversion ofR-(−)-N-(α-mercaptopropionyl)glycine andS-(+)-N-(α-mercaptopropionyl)glycine to each other have not occurred inbody. The specific experiment is a pharmacokinetic test in the body of atest animal, including determining the plasma drug concentration withhigh-performance liquid chromatography-mass spectrometry (HPLC-MS).

The process for treat a plasma sample to formulate an injected solutionincludes plasma acidification, extraction, and then derivatization, andis as follows:

1. plasma acidification, where the acid used for acidification can be,for example, hydrochloric acid, phosphoric acid, perchloric acid, oracetic acid, with hydrochloric acid being preferred, and hydrochloricacid of 1 mol/L being more preferred; and where the volume ratio of theacid to the plasma sample is (150 μl-250 μl):(2 ml-4 ml), with 200 μl:3ml being preferred;

2. extraction of the acidified plasma, where the organic solvent usedfor extraction can be, for example, ethyl acetate, chloroform,trichloromethane, diethyl ether, or n-hexane, with ethyl acetate beingpreferred; and

3. derivatization of the extract, where the useful derivatizationreagent can be, for example, phenyl isothiocyanate and2,3,4,6-tetra-O-acetyl-β-D-pyranoglucose isothiocyanate (GITC) solution,with GITC solution being preferred, GITC solution of 2 mg/ml being morepreferred, and solution of 2 mg/ml GITC in tetrahydrofuran being mostpreferred; where the derivatization temperature is 15-45° C., with25-35° C. being preferred, and 30° C. being more preferred; and wherethe derivatization time is 10-30 min, with 15-25 min being preferred,and 20 min being more preferred.

Chromatographic conditions include:

mobile phase A: methanol; mobile phase B: an aqueous solution,containing 0.05-0.20 mmol/L of sodium chloride and 5.0-6.0 mmol/L offormic acid, with 0.10 mmol/L of sodium chloride and 5.3 mmol/L offormic acid being preferred, where the ratio of A to B is (40-50:50-60,with 44:56 being preferred).

Mass spectrometric conditions include:

ionization mode: electrospray ionization; selective ion detection;curved desolvation line (CDL); temperature: 200° C.-300° C., with 250°C. being preferred; heating block temperature: 150° C.-250° C., with200° C. being preferred; CDL voltage: 20V-30V, with 25V being preferred;detection voltage: +1.2 kV-+1.8 kV, with +1.50 kV being preferred; flowrate of atomizing gas: 1.2 L/min-1.8 L/min, with 1.5 L/min beingpreferred; flow rate of drying gas: 1.5 L/min-2.5 L/min, with 2.0 L/minbeing preferred; detected ion: derivative of test drug [M+Na]+(m/z):575.20; internal standard: derivative of N-isobutanoyl-D-cysteine (NIDC)[M+Na]+(m/z): 603.05;

as long as the separation degree of the internal standard peak from themajor peak meets the requirements of Chinese Pharmacopoeia.

The results indicate that, in spectrogram ofR-(−)-N-(α-mercaptopropionyl)glycine at each time points after beingadministrated individually, the presence ofS-(+)-N-(α-mercaptopropionyl)glycine is not obviously detected;likewise, the presence of R-(−)-N-(α-mercaptopropionyl)glycine is notobviously detected either after administratingS-(+)-N-(α-mercaptopropionyl)glycine, which suggest that conversions toeach other have not occurred in body.

Therefore, it is practicable to make clinically useful pharmaceuticalpreparation from R-(−)-N-(α-mercaptopropionyl)glycine orS-(+)-N-(α-mercaptopropionyl)glycine.

In the present invention, the optically activeN-(α-mercaptopropionyl)glycine is made into a clinically usefulpharmaceutical preparation, which containsR-(−)-N-(α-mercaptopropionyl)glycine orS-(+)-N-(α-mercaptopropionyl)glycine, or a pharmaceutically acceptablesalt or ester thereof as active ingredient, and a pharmaceuticallyacceptable adjuvant, and is an orally administrated preparation or aninjection preparation.

The orally administrated preparation includes: a general oralpreparation, for example, tablet, capsule, granule, chewable tablet, oreffervescent tablet; a rapid release preparation, for example,dispersible tablet, or orally disintegrating tablet; or a slow releasepreparation, for example, slow release tablet, or slow release pellet.The injection preparation includes, for example, injectable solution,concentrated solution for injection, or sterile powder for injection.

The adjuvant in the oral pharmaceutical preparation includes a filler, abinder, or a disintegrant, in which the weight contents of the fillerand the disintegrant are 10-60%, and 2-30% respectively; and a glidant,a lubricant, and a surfactant can optionally exist, in which the weightcontents of the glidant, the lubricant, and the surfactant are 0.1-5%,0.1-5%, and 0.005-1% respectively. The filler can be starch,pregelatinized starch, carboxymethyl starch, microcrystalline cellulose,lactose, dextrin, sucrose, glucose, mannitol, sorbitol, calcium sulfatedihydrate, dibasic calcium phosphate, tribasic calcium phosphate, orcalcium carbonate. The binder can be corn starch, pregelatinized cornstarch, pregelatinized starch, gelatine, sucrose, arabic gum, povidone,methylcellulose of various viscosities, sodium carboxymethyl celluloseof low viscosity, ethylcellulose of various viscosities, polyvinylalcohol of various viscosities, polyethylene glycol 6000, or solution ofhydroxypropylmethyl cellulose in water or an alcohol. The disintegrantcan be starch, pregelatinized starch, low-substituted hydroxypropylcellulose, microcrystalline cellulose, absolute lignocellulose, alginicacid, sodium carboxymethyl starch, croscarmellose sodium, guar gum,crosslinked polyvinylpyrrolidone, ion exchange resin, methylcellulose,carboxymethylcellulose sodium, and effervescent disintegrant composed ofan organic acid (e.g., citric acid, and tartaric acid) and carbonate(e.g., sodium carbonate and sodium bicarbonate). The lubricant can betartaric acid, magnesium stearate, calcium stearate, zinc stearate,talc, polyethylene glycol 4000, polyethylene glycol 6000, polyethyleneglycol 8000, magnesium lauryl sulfate, sodium benzoate, sodium acetate,sodium chloride, sodium oleate, boric acid, leucine, adipic acid,fumaric acid, glycerol triacetate, polyoxyethylene monostearate,monolauryl saccharate, magnesium lauryl sulfate, and sodium laurylsulfate. The glidant can be gas phase micro-powder silica gel, syntheticmicro-powder silica gel, and magnesia. The surfactant can be sodiumdodecylsulfate, poloxamer, Tweens, Spans, hexadecyl trimethylaminebromide, sodium lauryl sulfate, sodium stearate sulfonate,polyoxyethylene castor oil, and polyoxyethylene monostearate. Theprescription can optionally contain flavor, including stevioside,fructose, sucrose, glucose, aspartame, protein sugar, xylitol, mannitol,sorbitol, lactose, maltitol, glycyrrhizin, sodiumcyclohexylaminosulfonate, banana flavor, orange flavor, pineappleflavor, mint flavor, fennel, vanillin, lemon flavor, cherry flavor, androse flavor. The prescription can also optionally contain wetting agent,i.e., aqueous or ethanol solution of different concentrations. Thecoating material useful in the pharmaceutical preparation of the presentinvention can be, for example, cellulose and derivatives thereof,acrylic resins, and polymers of ethylene.

The injection preparation includes injectable solution, concentratedsolution for injection, or sterile powder for injection. The adjuvant isan additive meeting injection requirements, including pH adjustingagent, isotonic adjustment agent, anti-oxidant, chelating agent, andexcipient in the sterile powder for injection. The pH adjusting agentincludes hydrochloric acid, lactic acid, methanesulfonic acid, sodiumhydroxide, sodium bicarbonate, phosphoric acid and salts thereof, aceticacid and salts thereof, citric acid and salts thereof, and amino acidand salts thereof; the isotonic adjustment agent includes glucose,sodium chloride, glycerol, and sodium sulfate; the excipient includessorbitol, mannitol, dextran, lactose, sucrose, glucose, hydrolyzedgelatine, and sodium chloride; the anti-oxidant includes 0.01%-0.1% ofsodium or potassium pyrosulfite, 0.01-0.5% of sodium sulfite, 0.01%-0.5%of sodium bisulphite, 0.01%-0.5% of sodium thiosulfate, 0.1-0.2% ofsodium formaldehydesulfoxylate, 0.05%-0.1% of thiourea, 0.05%-0.2% ofascorbic acid, 0.1%-0.5% of thioglycerol, 0.01-0.2% of glutathione,0.01%-0.5% of alanine, 0.01%-0.5% of cysteine, 0.01-0.1% of gallic acidand propyl or octyl ester thereof, 0.01-0.1% of tert-butylp-hydroxyanisole, 0.01%-0.5% of di-tert-butyl p-cresol, 0.01%-0.1% oftocopherol α, β, and γ, 0.01%-0.5% of nordihydroguaiaretic acid, or0.01%-0.5% of palmityl ascorbate; and the chelating agent includesdisodium ethylene diamine tetraacetate, and calcium sodium ethylenediamine tetraacetate.

The compound of the present invention is useful in preparation ofdetoxification medicament for improving metabolism.

The present invention further provides a liquid chromatography fordetermining an optical purity of an optically activeN-(α-mercaptopropionyl)glycine, including:

1) chromatographic conditions: the chromatography column has astationary phase of a chiral column with 3,5-dimethylphenyl-carbamateglycopeptides and a mobile phase of n-hexane/ethanol/glacial acetic acid(80-95:5-20:0.01-1.0), with (90:10:0.1) being preferred; the detectionwavelength is 200 nm-230 nm, with 210 nm being preferred; and flow rateof the mobile phase is 0.2-3.0 ml/min, with 1.0 ml/min being preferred;

2) formulation of the sample solution: the sampleR-(−)-N-(α-mercaptopropionyl)glycine orS-(+)-N-(α-mercaptopropionyl)glycine is formulated into a solution of0.1-20 mg/ml (preferably 1 mg/ml) with an organic solvent; and theorganic solvent is selected from n-propanol, i-propanol, ethanol,i-butanol, and methanol, with ethanol being preferred; and;

3) determination: the solution is injected into a high performanceliquid chromatograph (HPLC), and the chromatogram is recorded andanalyzed.

BRIEF DESCRIPTION OF THE DRAWINGS

No drawings.

DETAILED DESCRIPTION OF THE INVENTION Embodiment 1 Preparation ofR-(−)-N-(mercaptopropionyl)glycine

1) 54.3 g (0.5 mol) of R-(+)-2-chloropropionic acid, 60 g (0.504 mol) ofthionyl chloride were added to a 100 ml dry reaction flask, and stirredat reflux for 4 h with moisture isolated. After the reaction wascompleted, excessive thionyl chloride was distilled out for reuse, andthen the fraction having a by of 95-105° C. was collected, to get 55 gof R-(+)-2-chloropropionyl chloride as a colorless liquid with a yieldof 86.6%.

2) 29.9 g (0.40 mol) of glycine, 21.2 g (0.20 mol) of anhydrous sodiumcarbonate, and 250 ml of water were added into a 1000 ml reaction flask,and stirred until dissolved. Cooling with an ice-salt bath, 50.6 g (0.40mol) of R-(+)-2-chloropropionyl chloride was added dropwise withvigorously stirring, and a saturated solution of anhydrous sodiumcarbonate was added at the same time, to make the reaction solution weakbasic. After addition, stirring was continued for additional 3-5 h tillthe reaction was completed. The reaction solution was acidified to pH=1with concentrated hydrochloric acid, extracted with ethyl acetate, andthen dried over anhydrous magnesium sulfate. The reaction solution wasfiltered and concentrated under reduced pressure till a crystal wasprecipitated. Then, the reaction solution was stood still, filtered, anddried, to get 38.6 g of R-(+)-2-chloropropionyl glycine as white smallneedle crystal. mp: 120-124° C., [α]_(n) ²⁰=+23.8° C. (water).

3) 26.5 g (0.11 mol) of sodium sulfide (Na₂S.9H₂O), 3.52 g (0.11 mol) ofsublimed sulfur, and 120 ml of water were added to a 250 ml beaker, andheated with stirring until dissolved, to get a red brown solution ofsodium disulfide for later use. 16.4 g (0.10 mol) ofR-(+)-2-chloropropionyl glycine, and 5.6 g of anhydrous sodium carbonatewere added into a 250 ml reaction flask, and then 100 ml of water wasadded slowly to prevent the generation of bubbles. After cooling to0-10° C., the sodium disulfide solution was added dropwise, and thenreacted for 10-15 h at 5-15° C. After the reaction was completed, thereaction solution was cooled to about 0° C., to which concentratedsulfuric acid was added dropwise, to adjust pH to approximately 1. Then,the reaction solution was filtered, and then 17 g of zinc powder wasadded in portion to the filtrate with stirring, and reacted for 3 h atnormal temperature till the reaction was completed. The reactionsolution was filtered, and then the filtrate was extracted with ethylacetate, washed with saturated sodium chloride solution, and dried overanhydrous magnesium sulfate. The reaction solution was filtered,concentrated under reduced pressure, and stood still, to get a solid.The solid was filtered out and collected, and recrystallized with ethylacetate, and then dried under vacuum, to get 8.4 g ofR-(−)-N-(α-mercaptopropionyl)glycine as white crystalline solid. mp:102-104° C., [α]_(n) ²⁰=−36.5° C. (water), content: 99.3% (titrated with0.1 mol/L of iodine titration solution), related substance<2% (thinlayer chromatography: silica gel G thin plate,chloroform-acetone-glacial acetic acid (9:3:1), developed with iodinevapor).

1HNMR (DMSO-D6) δ ppm: 1.35 (d, 3H); 2.79 (d, 1H); 3.54 (m, 1H); 3.77(m, 2H); 8.25 (t, 1H); 12.5 (bs, 1H); MS (m/z) 163; element analysisC₅H₉NO₃S (%): C, 36.65; H, 5.66; N, 8.50; S, 19.68.

Embodiment 2 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine

1) 54.3 g (0.5 mol) of S-(−)-2-chloropropionic acid, 60 g (0.504 mol) ofthionyl chloride were added to a 100 ml dry reaction flask, and stirredat reflux for 4 h with moisture isolated. After the reaction wascompleted, excessive thionyl chloride was distilled out for reuse, andthen the fraction having a by of 95-105° C. was collected, to get 56 gof S-(−)-2-chloropropionyl chloride as a colorless liquid, with a yieldof 88.2%.

2) 29.9 g (0.40 mol) of glycine, 21.2 g (0.20 mol) of anhydrous sodiumcarbonate, and 250 ml of water were added into a 1000 ml reaction flask,and stirred until dissolved. Cooling with an ice-salt bath, 50.6 g (0.40mol) of S-(−)-2-chloropropionyl chloride was added dropwise withvigorously stirring, and a saturated solution of anhydrous sodiumcarbonate was added at the same time, to make the reaction solution weakbasic. After addition, stirring was continued for additional 3-5 h tillthe reaction was completed. The reaction solution was acidified to pH=1with concentrated hydrochloric acid, extracted with ethyl acetate, andthen dried over anhydrous magnesium sulfate. The reaction solution wasfiltered and concentrated under reduced pressure till a crystal wasprecipitated. Then, the reaction solution was stood still, filtered, anddried, to get 37.8 g of S-(−)-2-chloropropionyl glycine as white smallneedle crystal. mp: 120-124° C., [α]_(n) ²⁰=−24.2° C. (water).

3) 26.5 g (0.11 mol) of sodium sulfide (Na₂S.9H₂O), 3.52 g (0.11 mol) ofsublimed sulfur, and 120 ml of water were added to a 250 ml beaker, andheated with stirring until dissolved, to get a red brown solution ofsodium disulfide for later use. 16.4 g (0.10 mol) ofS-(−)-2-chloropropionyl glycine, and 5.6 g of anhydrous sodium carbonatewere added into a 250 ml reaction flask, and then 100 ml of water wasadded slowly to prevent the generation of bubbles. After cooling to0-10° C., the sodium disulfide solution was added dropwise, and thenreacted for 10-15 h at 5-15° C. After the reaction was completed, thereaction solution was cooled to about 0° C., to which concentratedsulfuric acid was added dropwise, to adjust pH to approximately 1. Thereaction solution was filtered, and then 17 g of zinc powder was addedin portion to the filtrate with stirring, and reacted for 3 h at normaltemperature till the reaction was completed. Then, the reaction solutionwas filtered, and then the filtrate was extracted with ethyl acetate,washed with saturated sodium chloride solution, and dried over anhydrousmagnesium sulfate. The reaction solution was filtered and concentratedunder reduced pressure, and stood still, to get a solid. The solid wasfiltered and collected, and recrystallized with ethyl acetate, and thendried under vacuum, to get 8.2 g of S-(+)-N-(α-mercaptopropionyl)glycineas white crystalline solid. mp: 102-104° C., [α]_(n) ²⁰=+37.5° C.(water), content: 99.1% (titrated with 0.1 mol/L of iodine titrationsolution), related substance<2% (thin layer chromatography: silica gel Gthin plate, chloroform-acetone-glacial acetic acid (9:3:1), developedwith iodine vapor).

1HNMR (DMSO-D6) δ ppm: 1.40 (d, 3H): 2.80 (d, 1H); 3.60 (m, 1H): 3.81(m, 2H); 8.28 (t, 1H); 12.8 (bs, 1H); MS (m/z) 163: element analysisC₅H₉NO₃S (%): C, 36.68; H, 5.65; N, 8.53; S, 19.65.

Embodiment 3 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine

1) 54.3 g (0.5 mol) of S-(−)-2-chloropropionic acid, 60 g (0.504 mol) ofthionyl chloride were added to a 100 ml dry reaction flask, and stirredat reflux for 4 h with moisture isolated. After the reaction wascompleted, excessive thionyl chloride was distilled out for reuse, andthen the fraction having a by of 95-105° C. was collected to get 54.6 gof S-(−)-2-chloropropionyl chloride as a colorless liquid.

2) 29.9 g (0.40 mol) of glycine, 21.2 g (0.20 mol) of anhydrous sodiumcarbonate, and 250 ml of water were added into a 1000 ml reaction flask,and stirred until dissolved. Cooling with an ice-salt bath, 50.6 g (0.40mol) of S-(−)-2-chloropropionyl chloride was added dropwise withvigorously stirring, and a saturated solution of anhydrous sodiumcarbonate was added at the same time, to make the reaction solution weakbasic. After addition, stirring was continued for additional 3-5 h tillthe reaction was completed. The reaction solution was acidified to pH=1with concentrated hydrochloric acid, extracted with ethyl acetate, andthen dried over anhydrous magnesium sulfate. The reaction solution wasfiltered and concentrated under reduced pressure till a crystal wasprecipitated. Then, the reaction solution was stood still, filtered, anddried, to get 38 g of S-(−)-2-chloropropionyl glycine as white smallneedle crystal.

3) 28.8 g (0.12 mol) of sodium sulfide (Na₂S.9H₂O), 3.84 g (0.12 mol) ofsublimed sulfur, and 120 ml of ethanol were added to a 250 ml beaker,and heated with stirring until dissolved, to get a red brown solution ofsodium disulfide for later use. 18.3 g of (0.11 mol) ofS-(−)-2-chloropropionyl glycine, and 6.3 g of anhydrous sodium carbonatewere added into a 250 ml reaction flask, and then 100 ml of water wasadded slowly to prevent the generation of bubbles. After cooling to0-10° C., the sodium disulfide solution was added dropwise, and thenreacted for 12 h at 0-10° C. After the reaction was completed, thetemperature was kept constant, and concentrated sulfuric acid was addeddropwise, to adjust pH to approximately 1. The reaction solution wasfiltered, and then 17 g of zinc powder was added in portion to thefiltrate with stirring, and reacted for 3 h at normal temperature tillthe reaction was completed. The reaction solution was filtered, and thenthe filtrate was extracted with ethyl acetate, washed with saturatedsodium chloride solution, and dried over anhydrous magnesium sulfate.The reaction solution was filtered and concentrated under reducedpressure, and stood still to get a solid. The solid was filtered,collected, recrystallized with ethyl acetate, and then dried undervacuum, to get 8.1 g of S-(+)-N-(α-mercaptopropionyl)glycine as whitecrystalline solid. Content: 99.3% (titrated with 0.1 mol/L of iodinetitration solution), related substance<2% (thin layer chromatography:silica gel G thin plate, chloroform-acetone-glacial acetic acid (9:3:1),developed with iodine vapor).

Embodiment 4 Determination of Optical Purity ofR-(−)-N-(α-mercaptopropionyl)glycine

Instrument: HPLC, SPD-10 Avp UV detector, LC-10AD pump

Mobile phase: n-hexane-ethanol-glacial acetic acid (90:10:0.1)

Flow rate: 1.0 ml/min

Chromatography column: CHIRALPAK AD-H (with 3,5-dimethylphenyl-carbamateglycopeptide as stationary phase)

Column temperature: 25° C.

Detection wavelength: 210 nm

Sample concentration: 0.5 mg/ml

The optical activity of R-(−)-N-(α-mercaptopropionyl)glycine obtained inEmbodiment 1 under the conditions above is determined to be 99.3%.

Embodiment 5 Determination of Optical Purity ofS-(+)-N-(α-mercaptopropionyl)glycine

Instrument: HPLC, SPD-10AvPUV detector, LC-10AD pump

Mobile phase: n-hexane-ethanol-glacial acetic acid (90:10:0.1)

Flow rate: 1.0 ml/min

Chromatography column: CHIRALPAKAD-H (with 3,5-dimethylphenyl-carbamateglycopeptide as stationary phase)

Column temperature: 25° C.

Detection wavelength: 210 nm

Sample concentration: 0.5 mg/ml

The optical activity of S-(+)-N-(α-mercaptopropionyl)glycine obtained inEmbodiment 1 under the conditions above is determined to be 99.2%.

Embodiment 6 Preparation of R-(−)-N-(α-mercaptopropionyl)glycineArginine Salt

10 g of R-(−)-N-(α-mercaptopropionyl)glycine obtained in Embodiment 1,11.2 g of L-arginine, and 60 ml of 95% methanol were added into areaction flask, stirred, heated to reflux, and reacted for 3 h. Afterthe reaction was completed, the reaction solution was filtered whilebeing hot, and then the filtrate was cooled to room temperature andplaced in a freezer to precipitate a crystal by cooling. The crystal wasfiltered, collected, and dried to obtain 19.2 g of a white crystallinesolid with a yield of 92.8%.

Embodiment 7 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine LysineSalt

10 g of S-(+)-N-(α-mercaptopropionyl)glycine obtained in Embodiment 2,9.4 g of L-lysine, and 60 ml of 95% methanol were added into a reactionflask, stirred, heated to reflux, and reacted for 3 h. After thereaction was completed, the reaction solution was filtered while beinghot, and then the filtrate was cooled to room temperature and placed ina freezer to precipitate a crystal by cooling. The crystal was filtered,collected, and dried to obtain 16.8 g of a white crystalline solid witha yield of 88.6%.

Embodiment 8 Preparation of R-(−)-N-(α-mercaptopropionyl)glycine sodium

1) 16.3 g (0.10 mol) of R-(−)—N(α-mercaptopropionyl)glycine, and 45 mlof methanol were placed in a reaction flask, to which 0.15 g ofdithiothreitol was added at 15° C., and stirred for 15 min, and then 5 gof molecular sieve was added.

2) 4.65 g of NaOH was slowly added into the methanol solution above in 6portions.

3) After addition, the reactants were reacted for 1 h at 15° C. withstirring, and then for another 1 h at 20-25° C. with stirring. After thereaction was completed, the molecular sieve was filtered off, and thenthe filtrate was added into 130 mL of acetone, and fully agitated to behomogeneously mixed.

4) The large amount of white precipitate present in the reactionsolution was filtered by suction, and then the filter cake was dried at80° C., to get 12.8 g of anhydrous R-(−)-N-(α-mercaptopropionyl)glycinesodium.

Embodiment 9 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine Sodium

1) 16.3 g (0.10 mol) of S-(+)-N-(α-mercaptopropionyl)glycine, and 45 mlof methanol were placed in a reaction flask, to which 0.15 g ofdithiothreitol was added at 15° C., and stirred for 15 min, and then 5 gof molecular sieve was added.

2) 4.65 g of NaOH was slowly added into the methanol solution above in 6portions.

3) After addition, the reactants were reacted for 1 h at 15° C. withstirring, and then for another 1 h at 20-25° C. with stirring. After thereaction was completed, the molecular sieve was filtered off, and thenthe filtrate was added into 130 mL of acetone, and fully agitated to behomogeneously mixed.

4) The large amount of white precipitate present in the reactionsolution was filtered by suction, and then the filter cake was dried at80° C., to get 13.1 g of anhydrous S-(+)-N-(α-mercaptopropionyl)glycinesodium.

Embodiment 10 Preparation of R-(−)-N-(α-mercaptopropionyl)glycine Tablet

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 100 g Microcrystalline cellulose170 g Pregelatinized starch  60 g 8% starch slurry suitable amountSodium carboxymethyl starch  7 g Magnesium stearate  3 g

Preparation process: Taking the preparation of 1000R-(−)-N-(α-mercaptopropionyl)glycine tablets as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofR-(−)-N-(α-mercaptopropionyl)glycine, microcrystalline cellulose, andpregelatinized starch were weighted, and homogeneously mixed to preparea soft material with 8% starch slurry, which was granulated with a sieveof 20 mesh, dried, and then sized with a sieve of 18 mesh; afterwards,formula amounts of sodium carboxymethyl starch and magnesium stearatewere added, homogeneously mixed, and then pressed into tablets. Theweight of each tablet is about 345 mg.

Embodiment 11 Preparation of Film-CoatedR-(−)-N-(α-mercaptopropionyl)glycine Tablet

The naked tablet obtained in Embodiment 10 was coated with a formulated8% gastric soluble OPADRY (OY-C-7000A) solution in ethanol in a highefficient coating pan; and the amount of the coating powder is 2.0-3.0%by weight of the naked tablet.

Embodiment 12 Preparation of Enteric SolubleR-(−)-N-(α-mercaptopropionyl)glycine Tablet

The naked tablet obtained in Embodiment 10 was coated with a formulated8% enteric soluble OPADRY solution in ethanol in a high efficientcoating pan; and the amount of the coating powder is 4.0-5.0% by weightof the naked tablet.

Embodiment 13 Preparation of Slow ReleaseR-(−)-N-(α-mercaptopropionyl)glycine Tablet

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 150 g Hydroxypropylmethyl cellulose(K15M) 150 g Lactose  50 g 3% povidone solution in 80% ethanol suitableamount Magnesium stearate  5 g

Preparation process: Taking the preparation of 1000 slow releaseR-(−)-N-(α-mercaptopropionyl)glycine tablets as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofR-(−)-N-(α-mercaptopropionyl)glycine, hydroxypropylmethyl cellulose, andlactose were weighted, and homogeneously mixed to prepare a softmaterial with 3% povidone solution in 80% ethanol, which was granulatedwith a sieve of 20 mesh, dried, and then sized with a sieve of 18 mesh;afterwards, formula amounts of magnesium stearate were added,homogeneously mixed, and then pressed into tablets. The weight of eachtablet is about 360 mg.

Embodiment 14 Preparation of R-(−)-N-(α-mercaptopropionyl)glycineDispersible Tablet

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 100 g  Crosslinkedpolyvinylpyrrolidone 15 g  Microcrystalline cellulose 180 g  Mannitol 50g  60% ethanol solution suitable amount Sodium dodecyl sulfate 0.2 g  Micro-powder silica gel 5 g Magnesium stearate 3 g Stevioside 5 g

Preparation process: Taking the preparation of 1000R-(−)-N-(α-mercaptopropionyl)glycine dispersible tablets as example, theraw and auxiliary materials were respectively pulverized and passedthrough a sieve of 100 mesh for later use; next, formula amounts ofR-(−)-N-(α-mercaptopropionyl)glycine, microcrystalline cellulose, andmannitol were weighted, and homogeneously mixed to prepare a softmaterial with 60% ethanol solution, which was granulated, dried, andthen sized; afterwards, formula amounts of crosslinkedpolyvinylpyrrolidone, sodium dodecyl sulfate, micro-powder silica gel,magnesium stearate, and stevioside were added, homogeneously mixed, andthen pressed into tablets. The weight of each tablet is about 360 mg.

Embodiment 15 Preparation of R-(−)-(α-mercaptopropionyl)glycine Capsule

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 150 g Starch  45 g 5% povidonesolution in 60% ethanol suitable amount Magnesium stearate  2 g

Preparation process: Taking the preparation of 1000R-(−)-N-(α-mercaptopropionyl)glycine capsules as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofR-(−)-N-(α-mercaptopropionyl)glycine, and starch were weighted, andhomogeneously mixed to prepare a soft material with 5% povidone solutionin 60% ethanol, which was granulated, dried, and then sized; afterwards,formula amounts of magnesium stearate was added, homogeneously mixed,and then filled into a capsule.

Embodiment 16 Preparation of R-(−)-N-(α-mercaptopropionyl)glycineGranule

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 100 g Mannitol 350 g Sucrose 350 gSodium carboxymethyl cellulose  50 g Aspartame  10 g Maltdextrin 140 g5% povidone solution in 80% ethanol suitable amount Flavor  1 g

Preparation process: Taking the preparation of 1000 packages ofR-(−)-N-(α-mercaptopropionyl)glycine granule as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, the flavor was added into 5%povidone solution in 80% ethanol, and then formula amounts ofR-(−)-N-(α-mercaptopropionyl)glycine, sucrose, mannitol, sodiumcarboxymethyl cellulose, aspartame, and maltodextrin were weighted toprepare a soft material with 5% povidone solution in 80% ethanol, whichwas granulated with a sieve of 16 mesh, dried, and then sized with asieve of 14 mesh; afterwards, the granulates were sieved to remove thefine powder with a sieve of 60 mesh, and packaged. The weight of eachpackage is about 1 g.

Embodiment 17 Preparation of InjectableR-(−)-N-(α-mercaptopropionyl)glycine Solution

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 100 g Water for injection made upto 2000 ml

Preparation process: Taking the preparation of 1000 ampoules ofinjectable R-(−)-N-(α-mercaptopropionyl)glycine solution as example,formula amount of R-(−)-N-(α-mercaptopropionyl)glycine was added into1600 ml of water for injection, and stirred until completely dissolved;then additional water for injection was added to make up to 2000 ml, anduniformly stirred; afterwards, 0.1% of activated carbon for refinementof injection was added, and uniformly stirred; the drug solution wasprocessed with titanium rod to remove active carbon, and then finelyfiltered with 0.45 μm and 0.22 μm micro membranes in sequence to asatisfactory clarification; then the solution was detected forintermediates, filled into 2 ml ampoules at 2 ml specification undernitrogen flow if eligible, and then sterilized.

Embodiment 18 Preparation of R-(−)-N-(α-mercaptopropionyl)glycineInjection

Formula Composition:

R-(−)-N-(α-mercaptopropionyl)glycine 100 g Dextran 40  50 g Sodiumbisulphate  1 g Disodium ethylenediamine tetraacetate  0.2 g

Preparation process: Taking the preparation of 1000 vials ofR-(−)-N-(α-mercaptopropionyl)glycine for injection as example, formulaamounts of R-(−)-N-(α-mercaptopropionyl)glycine, dextran 40, sodiumbisulphate, and disodium ethylenediamine tetraacetate were added into1600 ml of water for injection, and stirred until completely dissolved;next, the pH of the solution was adjusted to 1.5-2.5 with 1 mol/Lhydrochloric acid, and then additional water for injection was added tomake up to 2000 ml, and uniformly stirred; afterwards, 0.1% of activatedcarbon for refinement of injection was added, and uniformly stirred; thedrug solution was processed with titanium rod to remove active carbon,and then finely filtered with 0.45 μm and 0.22 μm micro membranes insequence and sterilized under aseptic conditions; the drug solution wasdetected for the presence of intermediates, pH, content, andclarification, and filled into a penicillin bottle at 2 ml specificationif eligible, partially stoppered, freeze dried, stoppered and capped,and then light inspected.

Embodiment 19 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine Tablet

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 100 g Microcrystalline cellulose170 g Pregelatinized starch  60 g 8% starch slurry suitable amountSodium carboxymethyl starch  7 g Magnesium stearate  3 g

Preparation process: Taking the preparation of 1000S-(+)-N-(α-mercaptopropionyl)glycine tablets as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofS-(+)-N-(α-mercaptopropionyl)glycine, microcrystalline cellulose, andpregelatinized starch were weighted, and homogeneously mixed to preparea soft material with 8% starch slurry, which was granulated with a sieveof 20 mesh, dried, and then sized with a sieve of 18 mesh; afterwards,formula amounts of sodium carboxymethyl starch and magnesium stearatewere added, homogeneously mixed, and then pressed into tablets. Theweight of each tablet is about 345 mg.

Embodiment 20 Preparation of Film-CoatedS-(+)-N-(α-mercaptopropionyl)glycine Tablet

The naked tablet obtained in Embodiment 19 was coated with a formulated8% gastric soluble OPADRY (OY-C-7000A) solution in ethanol in a highefficient coating pan; and the amount of the coating powder used is2.0-3.0% by weight of the naked tablet.

Embodiment 21 Preparation of Enteric SolubleS-(+)-N-(α-mercaptopropionyl)glycine Tablet

The naked tablet obtained in Embodiment 19 was coated with a formulated8% enteric soluble OPADRY solution in ethanol in a high efficientcoating pan; and the amount of the coating powder used is 4.0-5.0% byweight of the naked tablet.

Embodiment 22 Preparation of Slow ReleaseS-(+)-N-(α-mercaptopropionyl)glycine Tablet

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 150 g Hydroxypropylmethyl cellulose(K15M) 150 g Lactose  50 g 3% povidone solution in 80% ethanol suitableamount Magnesium stearate  5 g

Preparation process: Taking the preparation of 1000 slow releaseS-(+)-N-(α-mercaptopropionyl)glycine tablets as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofS-(+)-N-(α-mercaptopropionyl)glycine, hydroxypropylmethyl cellulose, andlactose were weighted, and homogeneously mixed to prepare a softmaterial with 3% povidone solution in 80% ethanol, which was granulatedwith a sieve of 20 mesh, dried, and then sized with a sieve of 18 mesh;afterwards, formula amount of magnesium stearate was added,homogeneously mixed, and then pressed into tablets. The weight of eachtablet is about 360 mg.

Embodiment 23 Preparation of S-(+)-N-(α-mercaptopropionyl)glycineDispersible Tablet

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 100 g  Crosslinkedpolyvinylpyrrolidone 15 g  Microcrystalline cellulose 80 g  Mannitol 50g  60% ethanol solution suitable amount Sodium dodecyl sulfate 0.2 g  Micro-powder silica gel 5 g Magnesium stearate 3 g Stevioside 5 g

Preparation process: Taking the preparation of 1000R-(−)-N-(α-mercaptopropionyl)glycine dispersible tablet as example, theraw and auxiliary materials were respectively pulverized and passedthrough a sieve of 100 mesh for later use; next, formula amounts ofS-(+)-N-(mercaptopropionyl)glycine, microcrystalline cellulose, andmannitol were weighted, and homogeneously mixed to prepare a softmaterial with 60% ethanol solution, which was granulated, dried, andthen sized; afterwards, formula amounts of crosslinkedpolyvinylpyrrolidone, sodium dodecyl sulfate, micro-powder silica gel,magnesium stearate, and stevioside were added, homogeneously mixed, andthen pressed into tablets. The weight of each tablet is about 360 mg.

Embodiment 24 Preparation of S-(+)-N-(α-mercaptopropionyl)glycineCapsule

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 150 g Starch  45 g 5% povidonesolution in 60% ethanol suitable amount Magnesium stearate  2 g

Preparation process: Taking the preparation of 1000S-(+)-N-(α-mercaptopropionyl)glycine capsules as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, formula amounts ofS-(+)-N-(α-mercaptopropionyl)glycine, and starch were weighted, andhomogeneously mixed to prepare a soft material with 5% povidone solutionin 60% ethanol, which was granulated, dried, and then sized; afterwards,formula amounts of magnesium stearate was added, homogeneously mixed,and then filled into a capsule.

Embodiment 25 Preparation of S-(+)-N-(α-mercaptopropionyl)glycineGranule

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 100 g Mannitol 350 g Sucrose 350 gCarboxymethylcellulose sodium  50 g Aspartame  10 g Maltdextrin 140 g 5%povidone solution in 80% ethanol suitable amount Flavor  1 g

Preparation process: Taking the preparation of 1000 packages ofS-(+)-N-(α-mercaptopropionyl)glycine granule as example, the raw andauxiliary materials were respectively pulverized and passed through asieve of 100 mesh for later use; next, the flavor was added into 5%povidone solution in 80% ethanol, and then, formula amounts ofS-(+)-N-(α-mercaptopropionyl)glycine, mannitol, sucrose, sodiumcarboxymethyl cellulose, aspartame, and maltodextrin were weighted toprepare a soft material with 5% povidone solution in 80% ethanol, whichwas granulated with a sieve of 16 mesh, dried, and then sized with asieve of 14 mesh; afterwards, it was sieved to remove the fine powderwith a sieve of 60 mesh, and packaged. The weight of each package isabout 1 g.

Embodiment 26 Preparation of InjectableS-(+)-N-(α-mercaptopropionyl)glycine Solution

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 100 g Sodium pyrosulfite 1 gDisodium ethylene diamine tetraacetate 0.2 g Water for injection made upto 2000 ml

Preparation process: Taking the preparation of 1000 ampoules ofinjectable S-(+)-N-(α-mercaptopropionyl)glycine solution as example,formula amounts of S-(+)-N-(α-mercaptopropionyl)glycine, sodiumpyrosulfite and disodium ethylenediamine tetraacetate were added into1600 ml of water for injection, and stirred until completely dissolved;then additional water for injection was added to make up to 2000 ml, anduniformly stirred; afterwards, 0.1% of activated carbon for refinementof injection was added, and uniformly stirred; the drug solution wasprocessed with titanium rod to remove active carbon, and then finelyfiltered with 0.45 μm and 0.22 μm micro membranes in sequence to asatisfactory clarification; then the solution was detected forintermediates, filled into 2 ml ampoules at 2 ml specification undernitrogen flow if eligible, and then sterilized.

Embodiment 27 Preparation of S-(+)-N-(α-mercaptopropionyl)glycine forInjection

Formula Composition:

S-(+)-N-(α-mercaptopropionyl)glycine 100 g Dextran 40  50 g

Preparation process: Taking the preparation of 1000 vials ofS-(+)-N-(α-mercaptopropionyl)glycine for injection as example, formulaamounts of S-(+)-N-(α-mercaptopropionyl)glycine, and dextran 40 wereadded into 1600 ml of water for injection, and stirred until completelydissolved; next, the pH of the solution was adjusted to 1.5-2.5 with 1mol/L hydrochloric acid or 1 mol/L sodium hydroxide solution, and thenadditional water for injection was added to make up to 2000 ml, anduniformly stirred; afterwards, 0.1% of activated carbon for refinementof injection was added, and uniformly stirred; the drug solution wasprocessed with titanium rod to remove active carbon, and then finelyfiltered with 0.45 μm and 0.22 μm micro membranes in sequence andsterilized under aseptic conditions; the solution was detected for thepresence of intermediates, pH, content, and clarification, and filledinto a penicillin at 2 ml specification if eligible, partiallystoppered, freeze dried, stoppered and capped, and then light inspected.

Embodiment 28 Effect of Optically Active N-(α-mercaptopropionyl)glycineAdministrated by Intravenous Injection on Serum Biochemical Indicatorsand Liver Indexes of Mice with Liver Injury Induced by Acetamidophenol

The animals were randomly divided into the following 5 groups of 10animals according to weight: solvent control group, model group(acetamidophenol, 400 mg/kg, ml/kg, intraperitoneal injection),tiopronin group (MPG), R-(−)-N-(α-mercaptopropionyl)glycine group, andS-(+)-N-(α-mercaptopropionyl)glycine group. After fasting for 12 h,except the solvent control group and model group were given saline byintravenous injection, the other test groups were intravenously injecteddrugs, and then intraperitoneally injected 400/mg/kg of acetamidophenolimmediately, to induce acute liver cell injury. The animals were fedwith food at 6 hours after modeling, and then fasted for 16 hours; andat 24 hours after modeling, blood were sampled by eye ball removal,centrifuged, and collected for serum. ALT, and AST were determinedfollowing the method of the kit. 10% of liver homogenate was prepared,to measure GSH. The liver and spleen were taken at the same time,weighted, and calculated for organcoefficient. The results are shown inTables 1 and 2.

TABLE 1 Effect of MPG and optical isomer thereof administrated byintravenous injection on serum biochemical indicators of mice with liverinjury induced by acetamidophenol Serum Number Liver ALT AST totalDosage of coefficient (Karl-Fischer (Karl-Fischer protein Group (mg/kg)animals (mg/g) unit) unit) (mg/ml) Solvent control group — 12 5.405 ±0.508 30.5 ± 18.1 30.0 ± 6.5  47.5 ± 4.7 Model group — 10 6.139 ± 0.6835761.8 ± 2761.8 1682.8 ± 1122.0 42.9 ± 8.1 Tiopronin group 800 8 5.804 ±0.598 648.1 ± 412.1 Not detected Not detected due to due to insufficientinsufficient serum serum S-(+)-N-(α-mercaptopropionyl)glycine 800 126.201 ± 0.567 2880.2 ± 2578.5 1201.2 ± 1123.0 43.7 ± 5.7 groupR-(−)-N-(α-mercaptopropionyl)glycine 800 12 5.816 ± 0.613 1812.1 ±1780.1 591.3 ± 528.0 43.8 ± 7.4 group *P < 005, **P < 0.01 compared withthe model group

It can be known from Table 1 that, compared with the solvent controlgroup, the liver coefficient of the mice in the model group issignificantly increased, and inhibition of the elevation of serum ALT isapparent in all the dosed groups, in which the function in theR-(−)-N-(α-mercaptopropionyl)glycine group is higher than that in theS—H—N-(α-mercaptopropionyl)glycine group. The ALT value of tioproningroup is lower than that of R-(−)-N-(α-mercaptopropionyl)glycine group,but in the experiment, after modeling, 4 mouse dead in the tioproningroup die, 2 mouse dead in the model group, and no death occurs in theR-(−)-N-(α-mercaptopropionyl)glycine group. Therefore, the protectionagainst liver injury in mice induced by acetamidophenol inR-(−)-N-(α-mercaptopropionyl)glycine group is superior to that intiopronin group.

TABLE 2 Effect of MPG and optical isomer thereof administrated byintravenous injection on liver indexes of mice with liver injury inducedby acetamidophenol Liver MDA Number total Liver (nmol/g Dosage ofprotein albumin GSH wet Group (mg/kg) animal (mg/ml) (mg/ml) (mo1/L)weight) Solvent control group — 12 17.4 ± 3.6 10.0 ± 1.9  1.20 ± 0.15288.9 ± 84.5 Model group — 10 13.0 ± 1.5 8.3 ± 0.7 0.93 ± 0.12  452.2 ±132.8 Tiopronin group 800 8 15.7 ± 1.4 9.6 ± 1.1 0.99 ± 0.16 362.4 ±98.6 S-(+)-N-(α-mercaptopropionyl)glycine 800 12 16.2 ± 2.9 9.1 ± 2.00.99 ± 0.16  440.0 ± 134.6 group R-(−)-N-(α-mercaptopropionyl)glycine800 12 16.5 ± 1.7 9.2 ± 1.3 1.11 ± 0.16 321.7 ± 72.9 group *P < 0.05,**P < 0.01 compared with the model group

It can be known from Table 2 that, compared with the model group, thecontent of liver total protein is significantly increased in all thedosed groups, liver GSH content is obviously increased and MDA contentis decreased in the R-(−)-N-(α-mercaptopropionyl)glycine group, but nosuch effects are present in the S—H—N-(α-mercaptopropionyl)glycine groupand tiopronin group. The results indicate thatR-(−)-N-(α-mercaptopropionyl)glycine can provide better protectionagainst liver injury.

Embodiment 29 Protection of N-(α-mercaptopropionyl)glycine and OpticalIsomer Thereof Against Liver Injury in Rats Induced by CarbonTetrachloride

The animals were randomly divided into the following 5 groups of 10animals according to weight: solvent control group, model group (50%CCl₄, 2 ml/kg, intraperitoneal injection), tiopronin group (MPG),R-(−)-N-(α-mercaptopropionyl)glycine group, andS-(+)-N-(α-mercaptopropionyl)glycine group. Except the solvent controlgroup was given olive oil by intraperitoneal injection, the other testgroups were intragastrically administrated drugs for 4 days, once daily,intraperitoneally injected CCl₄ once on the fifth day to induce acuteliver cell injury, treated with drugs 30 min before modeling (except forthe model control group), and then administrated drugs once 2 hoursafter modeling. Then, the animals were fasted for 16 h, blood wassampled from retrobulbar venous plexus 24 hours after injecting CCl₄,centrifuged, and collected for serum, and ALT, AST, albumin weredetermined following the method of the kit. The results are shown inTables 3, and 4. The left lobe of liver was taken to prepare 10% ofliver homogenate and measure GSH and MDA contents. The results are shownin Table 5.

TABLE 3 Effects of MPG and optical isomer thereof on ALT, and AST ofrats with liver injury induced by CCl₄ Dosage ALT (Karl-Fischer AST(Karl-Fischer Group (mg/kg, ig) unit) unit) Solvent control group —327.6 ± 11.4  117.6 ± 46.5 Model group — 1020.1 ± 275.0  1448.1 ± 362.5Tiopronin group 450 748.6 ± 260.2 1126.0 ± 159.2S-(+)-N-(α-mercaptopropionyl)glycine 450 532.8 ± 352.7  818.3 ± 347.0group R-(−)-N-(α-mercaptopropionyl)glycine 450 432.8 ± 252.6  718.7 ±336.2 group *P < 0.05, **P < 0.01 compared with the model group

It can be known from Table 3 that, all the three drugs can significantlydecrease the ALT and AST level, in which the decrease in theS-(+)-N-(α-mercaptopropionyl)glycine group and theR-(−)-N-(α-mercaptopropionyl)glycine group is higher than that in thetiopronin group.

TABLE 4 Effects of MPG and optical isomer thereof on serum proteincontent of rats with liver injury induced by CCl₄ Dosage Total (mg/kg,protein Albumin Globulin Group ig) (mg/ml) (mg/ml) (mg/ml) A/G Solventcontrol group — 76.2 ± 5.14 30.1 ± 2.23 46.1 ± 5.13 0.660 ± 0.087 Modelgroup — 59.1 ± 7.56 26.8 ± 2.74 32.3 ± 4.99 0.837 ± 0.059 Tioproningroup 450 59.8 ± 6.26 26.8 ± 1.98 32.9 ± 4.79 0.825 ± 0.101S-(+)-N-(α-mercaptopropionyl)glycine 450 64.7 ± 6.40 27.5 ± 1.44 37.2 ±5.06 0.747 ± 0.073 group R-(−)-N-(α-mercaptopropionyl)glycine 450 70.6 ±3.63 30.3 ± 1.20 40.3 ± 2.98 0.754 ± 0.050 group **P < 0.01 comparedwith the model group

It can be known from Table 4 that, compared with the model group, in theR-(−)-N-(α-mercaptopropionyl)glycine group, the serum total protein,albumin and globulin contents are obviously increased, and A/G value isdecreased; A/G value is also decreased in theS-(+)-N-(α-mercaptopropionyl)glycine group; but no obvious effect on theindexes are detected in the tiopronin group, which corresponds to theresults obtained in the model group.

1-12. (canceled)
 13. An optically active N-(α-mercaptopropionyl)glycine,being levo or dextro N-(α-mercaptopropionyl)glycine; wherein: the levoN-(α-mercaptopropionyl)glycine is S-(−)-N-(α-mercaptopropionyl)glycine:

of S-configuration, or a pharmaceutically acceptable salt or esterthereof; and the dextro N-(α-mercaptopropionyl)glycine isR-(+)-N-(α-mercaptopropionyl)glycine:

of R-configuration, or a pharmaceutically acceptable salt or esterthereof.
 14. The optically active N-(α-mercaptopropionyl)glycineaccording to claim 13, wherein the salt is an amino acid salt or a metalsalt, the amino acid salt has a general structural formula below:

wherein R is an amino acid selected from arginine, lysine, glycine,aspartic acid, alanine, phenylalanine, leucine, isoleucine, ornithine,cystine, cysteine, tyrosine, valine, serine, histidine, threonine,tryptoophan, methionine, methionine, proline, glutamic acid, orhydroxyproline; and the metal salt has a general structural formulabelow:

wherein R₀ is potassium, or sodium.
 15. The optically activeN-(α-mercaptopropionyl)glycine according to claim 13, wherein the esterhas a general structural formula below:

wherein R₁ is a linear C₁-C₅ alkyl group.
 16. A method for preparing theoptically active N-(α-mercaptopropionyl)glycine according to claim 13,comprising: reacting R-(+)-2-chloropropionic acid with thionyl chlorideto get R-(+)-2-chloropropionyl chloride; next, reactingR-(+)-2-chloropropionyl chloride with glycine under a weak basiccondition to get R-(+)-2-chloropropionyl glycine; and then, reactingR-(+)-2-chloropropionyl glycine with sodium disulfide to getS-(−)-N-(α-mercaptopropionyl)glycine; or comprising: reactingS-(−)-2-chloropropionic acid with thionyl chloride to getS-(−)-2-chloropropionyl chloride; next, reacting S-(−)-2-chloropropionylchloride with glycine under a weak basic condition to getS-(−)-2-chloropropionyl glycine; and then, reactingS-(−)-2-chloropropionyl glycine with sodium disulfide to getR-(+)-N-(α-mercaptopropionyl)glycine; wherein the sodium disulfide isprepared by reacting sodium sulfide with sublimed sulfur.
 17. Apharmaceutical preparation comprising the optically activeN-(α-mercaptopropionyl)glycine according to claim 13, or apharmaceutically acceptable salt or ester thereof.
 18. Thepharmaceutical preparation according to claim 17, wherein the medicamentcomprising S-(−)-N-(α-mercaptopropionyl)glycine orR-(+)-N-(α-mercaptopropionyl)glycine, or a pharmaceutically acceptablesalt or ester thereof as active ingredient, and a pharmaceuticallyacceptable adjuvant.
 19. The pharmaceutical preparation according toclaim 17, wherein the preparation is an orally administratedpreparation, comprising a general oral preparation, a rapid release oralpreparation, or a slow release preparation, and the general oralpreparation comprises tablet, capsule, granule, chewable tablet, oreffervescent tablet, the rapid release oral preparation is dispersibletablet, or orally disintegrating tablet, and the slow releasepreparation is slow release tablet or slow release pellet.
 20. Thepharmaceutical preparation according to claim 17, wherein thepreparation is an injection preparation, comprising injectable solution,concentrated solution for injection, or sterile powder for injection.21. The pharmaceutical preparation according to claim 18, wherein theadjuvant in the oral pharmaceutical preparation comprises a filler, abinder, or a disintegrant, wherein the weight contents of the filler andthe disintegrant are 10-60%, and 2-30% respectively; and a glidant, alubricant, and/or a surfactant optionally, wherein the weight contentsof the glidant, the lubricant, and the surfactant are 0.1-5%, 0.1-5%,and 0.005-1% respectively.
 22. The pharmaceutical preparation accordingto claim 19, wherein the adjuvant in the oral pharmaceutical preparationcomprises a filler, a binder, or a disintegrant, wherein the weightcontents of the filler and the disintegrant are 10-60%, and 2-30%respectively; and a glidant, a lubricant, and/or a surfactantoptionally, wherein the weight contents of the glidant, the lubricant,and the surfactant are 0.1-5%, 0.1-5%, and 0.005-1% respectively. 23.The pharmaceutical preparation according to claim 18, wherein theadjuvant in the injection preparation is an additive meeting injectionrequirements, comprising a pH adjusting agent, an isotonic adjustmentagent, an anti-oxidant, a chelating agent, and/or an excipient.
 24. Thepharmaceutical preparation according to claim 19, wherein the adjuvantin the injection preparation is an additive meeting injectionrequirements, comprising a pH adjusting agent, an isotonic adjustmentagent, an anti-oxidant, a chelating agent, and/or an excipient.
 25. Amethod for treating acute and chronic liver diseases, peripheral bloodleukopenia caused by chemoradiation therapy, early senile cataract, orvitreous opacities, wherein the acute and chronic liver diseases includeacute and chronic hepatitis, viral hepatitis, alcoholic hepatitis ordrug induced hepatitis, heavy metal toxic hepatitis, fatty liver, acuteand chronic liver injury, and cirrhosis, comprising orally administeringor injecting a pharmaceutical preparation according to claim
 17. 26. Aliquid chromatography for determining an optical purity of an opticallyactive N-(α-mercaptopropionyl)glycine, comprising: (1) chromatographicconditions: a chromatography column is a chiral column with3,5-dimethylphenyl-carbamate glycopeptide as stationary phase andn-hexane/ethanol/glacial acetic acid (80-95:5-20:0.01-1.0) as mobilephase; the detection wavelength is 200 nm-230 nm; and the flow rate ofmobile phase is 0.2-3.0 ml/min; (2) formulation of sample solution:sample S-(−)-N-(α-mercaptopropionyl)glycine orR-(+)-N-(α-mercaptopropionyl)glycine is formulated in an organic solventto give a solution of 0.1-20 mg/ml; wherein the organic solvent isselected from n-propanol, i-propanol, ethanol, i-butanol, and methanol;and (3) determination: the solution is injected into a high performanceliquid chromatograph (HPLC), the chromatogram is recorded and analyzed.27. The liquid chromatography according to claim 26, wherein then-hexane/ethanol/glacial acetic acid ratio is 90:10:0.1.
 28. The liquidchromatography according to claim 26, wherein the detection wavelengthis 210 nm.
 29. The liquid chromatography according to claim 26, whereinthe flow rate of the mobile phase is 1.0 ml/min.
 30. The liquidchromatography according to claim 26, wherein the organic solvent isethanol.
 31. A method for determining a pharmacokinetic profile of anoptically active N-(α-mercaptopropionyl)glycine, comprising: determiningthe plasma drug concentration in a test animal with high performanceliquid chromatography-mass spectrometry (HPLC-MS), wherein: a processfor treating a plasma sample to formulate an injected solution comprisesplasma acidification, extraction, and then derivatization, and is asfollows:
 1. plasma acidification, wherein the acid used foracidification comprises hydrochloric acid, phosphoric acid, perchloricacid, or acetic acid; and wherein the volume ratio of the acid to theplasma sample is (150 μl-250 μl):(2 ml-4 ml);
 2. extraction of theacidified plasma, wherein the organic solvent used for extractioncomprises ethyl acetate, chloroform, trichloromethane, diethyl ether, orn-hexane; and
 3. derivatization of the extract, wherein the usefulderivatization reagent comprises phenyl isothiocyanate and2,3,4,6-tetra-O-acetyl-β-D-pyranoglucose isothiocyanate (GITC) solution;wherein the derivatization temperature is 15-45° C.; and where thederivatization time is 10-30 min; chromatographic conditions comprise:mobile phase A: methanol; mobile phase B: an aqueous solution,containing 0.05-0.20 mmol/L of sodium chloride and 5.0-6.0 mmol/L offormic acid, wherein the ratio of A to B is (40-50:50-60); massspectrometric conditions comprise: ionization mode: electrosprayionization; selective ion detection; curved desolvation line (CDL);temperature: 200° C.-300° C.; heating block temperature: 150° C.-250°C.; CDL voltage: 20V-30V; detection voltage: +1.2 kV-+1.8 kV; flow rateof atomizing gas: 1.2 L/min-1.8 L/min; flow rate of drying gas: 1.5L/min-2.5 L/min; detected ion: derivative of test drug [M+Na]+(m/z):575.20; internal standard: derivative of N-isobutanoyl-D-cysteine (NIDC)[M+Na]+(m/z): 603.05; as long as the separation degree of the internalstandard peak from the major peak meets the requirements of ChinesePharmacopoeia.
 32. The method according to claim 31, wherein the acidused for acidification comprises hydrochloric acid.